Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays

Molecular biology has become an integral part of the diagnosis of infectious diseases. Recently, quantitative real-time PCR (QPCR) methods (often in the form of so-called TaqMan┬« systems) have been developed for the diagnosis of a wide range of infectious diseases; these techniques found valuable clinical application in the diagnosis and evaluation of progress and therapeutic success of viral diseases…

Contents

Introduction
Background
Virus structure and classification
RNA viruses
Replication strategies of RNA viruses
Conserved secondary structures in viral RNA genomes
Pathogenicity and virulence of RNA viruses
Transmission of enveloped RNA viruses
RNA viruses as causes of zoonoses
Human RNA viruses in the present work
The retrovirus family (Retroviridae)
Human endogenous retroviruses (HERVs)
Mouse mammary tumour virus (MMTV) and related retroviruses
Influenza viruses, the Orthomyxoviridae
Human and animal coronaviruses, Coronaviridae
Diagnosis of viral diseases
Aims
Goal and specific aims
Specific aims
Materials and Methods
Materials
Clinical specimens
Papers
Template handling and preparation
Methods
Development of sequence-based diagnostic tool
Principle of PCR/Real-Time PCR
TaqMan
 chemistry
SYBRGreen chemistry
Reverse transcription polymerase chain reaction (RT-PCR)
Broadly targeted QPCR
Design of degenerated primers
Design of degenerated probes
Introduction of LNATM residues to enhance Tm
Novel probes: Triple probe and MegaBeacon
Triple-probe concept
MegaBeacon probe
Comparison of MegB and conventional MB
Further PCR optimization
Search for representative sequences
Optimisation of annealing temperature
Multiplex QRT-PC…
….

Author: Muradrasoli, Shaman

Source: Uppsala University Library

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